Hydrogen-producing bacterium, Clostridium perfringens

ABSTRACT

The present invention aims to provide a hydrogen-producing bacterium, which excels in hydrogen yield and hydrogen production rate, and is usable for industrial hydrogen production from biomass as a production source. That is, it is intended to provide a bacterium belonging to the genus  Clostridium  which has a property of producing hydrogen at a rate of 60 mmol or more per hour per liter of a culture liquid which contains glucose as a substrate, by batch cultivation in a YNU anaerobic culture medium at 47° C. and pH 6.0, and a hydrogen production method comprising the use of the bacterium belonging to the genus  Clostridium.

TECHNICAL FIELD

The present invention relates to a novel hydrogen-producing bacteriumbelonging to the genus Clostridium, and a hydrogen production methodusing the hydrogen-producing bacterium.

BACKGROUND ART

In recent years, hydrogen fuel has been attracting attention as asubstitute energy for fossil fuels such as petroleum, because it is arenewable and clean energy which, unlike fossil fuels, emits littlecarbon dioxide and other environmental pollutants by burning. For thisreason, more efficient hydrogen production methods have beenenthusiastically studied all over the world.

Hydrogen can be produced from various production sources, although it ispreferably produced from biomass as a production source from a viewpointof recycling. Such hydrogen production methods from biomass mainlyinvolve thermochemical methods and biological methods with use ofbacteria. Of these, biological methods are preferred. The reason is thatthermochemical methods such as hot gasification are cost-consuming sincebiomass normally contains lots of moisture because of its nature as anenergy product of organic wastes, sugar cane, etc.

Bacteria for use in hydrogen production from biomass can be largelycategorized in photosynthetic bacteria and fermentative bacteria. Thephotosynthetic bacteria are capable of complete decomposition of organicmatters contained in biomass into water and carbonic gas by using lightenergy. However, the drawback is that light energy can be used onlyduring the day and becomes insufficient in the morning and the evening.On the other hand, the fermentative bacteria are capable of producinghydrogen even in a sealed container, and are suitable for hydrogenproduction from biomass having high moisture contents such as rawgarbage and waste molasses. In addition, the hydrogen production rate offermentative bacteria is higher than that of photosynthetic bacteria.For these reasons, hydrogen production methods with use of fermentativebacteria have been mainly employed in the field of industry.

Anaerobic fermentative bacteria for use in the hydrogen production frombiomass include bacteria belonging to the genus Clostridium such asClostridium butyricum and bacteria belonging to the genus Enterobactersuch as Enterobacter aerogenes (for example, refer to Non-patentDocument 1). As a hydrogen production method from biomass with use of abacterium belonging to the genus Clostridium, for example, there isdisclosed (1) a method for production of hydrogen by generating hydrogenfrom an organic material, comprising: a charging step for charging theorganic material; a microorganism-charging step for charging amicroorganism belonging to the genus Clostridium; a reaction step forreacting the microorganism with the organic material to producehydrogen; and a stirring step, during the reaction step, for stirringthe organic material and the microorganism in order to promote thereaction and decomposition (for example, refer to Patent Document 1). Inaddition, as a hydrogen production method from biomass with use of abacterium belonging to the genus Enterobacter, for example, there isdisclosed (2) a method for producing hydrogen and ethanol from a rawmaterial liquid which contains a biodiesel waste liquid obtained throughdemethylesterification of a methylesterified-oil and fat wherein themethod comprises a fermentation step for fermenting with a bacteriumbelonging to the genus Enterobacter at least in the presence of acarrier whose surface is capable of immobilizing microorganisms (forexample, refer to Patent Document 2).

Bacteria belonging to the genus Clostridium and bacteria belonging tothe genus Enterobacter are normally hydrogen-producing bacteria whichproduce hydrogen at 30 to 38° C. In contrast, there existhigh-temperature hydrogen-producing bacteria which produce hydrogen at65 to 80° C., and high-temperature hydrogen fermentation methods usingthe same. As compared to the hydrogen production methods using bacteriabelonging to the genus Clostridium or the like, the high-temperaturehydrogen fermentation methods are advantageous in the points of improvedhydrogen yield and easy prevention of the substrate contamination causedby various germs. For example, Thermotoga maritima has achieved atheoretically maximum hydrogen yield of 4 mol/mol-glucose by batchcultivation at 80° C. (for example, refer to Non-patent Document 2).Such a high hydrogen yield at high temperature can be attributed to thesuppression against metabolism of undesirable by-products such as lacticacid.

Non-patent Document 1: de Vrije and Classen (2003) “Dark hydrogenfermentations” in Bio-methane and Bio-hydrogen, p103 to 121

Patent Document 1: Japanese Unexamined Patent Application, FirstPublication No. 2001-157595

Patent Document 2: Japanese Unexamined Patent Application, FirstPublication No. 2006-180782

Non-patent Document 2: Schroder et al. (1994) Archives of microbiology161: p460 to 470

DISCLOSURE OF INVENTION

Biomass such as a food waste etc. contains various substances whichinhibit hydrogen-producing bacteria from producing hydrogen. Therefore,hydrogen-producing bacterium for use in hydrogen production from biomassdesirably excel in hydrogen yield and hydrogen production rate.

However, known bacteria belonging to the genus Enterobacter and the likewhich produce hydrogen at 30 to 38° C. are not sufficient in hydrogenyield and hydrogen production rate, and it is very difficult to use suchbacteria for industrial hydrogen production from biomass as a productionsource.

On the other hand, high-temperature hydrogen-producing bacteria whichproduce hydrogen at 65 to 80° C. are very inferior in hydrogenproduction rate although they excel in hydrogen yield. Therefore, thesebacteria are not suitable for industrial use, either. For example, thehydrogen production rate of Thermotoga maritima is no more than 10mmol/L·h per hour per liter of a batch culture liquid at 80° C. (forexample, refer to Non-patent Document 2). Such a low hydrogen productionrate at high temperature can be attributed to a low cell density of thebacterium.

The present invention is intended to provide a hydrogen-producingbacterium, which excels in hydrogen yield and hydrogen production rate,and is usable for industrial hydrogen production from biomass as aproduction source.

In addition, the present invention is also intended to provide ahydrogen production method which uses the hydrogen-producing bacterium.

The inventors of the present invention have conducted intensive studiesto solve the abovementioned problems. As a result, they have come to theopinion that both the hydrogen yield and the hydrogen production ratecan be improved by producing hydrogen at an intermediate temperaturebetween 30 to 38° C. and 65 to 80° C., and have singled out ahydrogen-producing bacterium capable of satisfactorily producinghydrogen at about 50° C. This has led to the completion of the presentinvention.

That is, the present invention relates to the following aspects.

(1) A bacterium belonging to the genus Clostridium which has a propertyof producing hydrogen at a rate of 60 mmol or more per hour per liter ofa culture liquid which contains glucose as a substrate, by cultivationin a YNU anaerobic culture medium at 47° C. and pH 6.0.

(2) A bacterium belonging to the genus Clostridium according to (1),wherein the bacterium is Clostridium perfringens.

(3) A bacterium belonging to the genus Clostridium according to eitherone of (1) and (2), wherein the optimum temperature for hydrogenproduction is 47 to 50° C.

(4) A bacterium belonging to the genus Clostridium according to any oneof (1) through (3), wherein the optimum temperature for cell growth is44 to 47° C.

(5) A bacterium belonging to the genus Clostridium according to any oneof (1) through (4), wherein the bacterium has a raffinose-decomposingability.

(6) A bacterium belonging to the genus Clostridium according to any oneof (1) through (5), wherein the bacterial strain is Clostridiumperfringens HN001 (NITE BP-318).

(7) A hydrogen production method comprising the use of the bacteriumbelonging to genus Clostridium according to any one of (1) through (6).

In the hydrogen production method according to (7), the bacteriumbelonging to genus Clostridium is preferably cultured at 47 to 50° C. Inaddition, the pH of the culture liquid used therein is preferably 5.8 to6.5, and more preferably 6.0 to 6.2.

(8) Use of the bacterium belonging to genus Clostridium according to anyone of (1) through (6) for the production of hydrogen.

The bacterium belonging to the genus Clostridium of the presentinvention is a hydrogen-producing bacterium which is not inferior inhydrogen yield and quite excels in hydrogen production rate.Accordingly, the use of the bacterium belonging to the genus Clostridiumof the present invention and the hydrogen production method of thepresent invention are capable of hydrogen production with higherefficiency than ever before, even from biomass as a production source.In addition, the present invention is capable of maintaining thetemperature with less energy as compared to high-temperature hydrogenfermentation methods which require a large amount of energy formaintaining the temperature, and thus is preferable in terms of economy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the effect of the temperature on the growth of Clostridiumperfringens strain HN001. FIG. 1A shows the measurement results oftimewise changes in the bacterial dry weight at respective temperatures,wherein the Y axis shows the bacterial dry weight per liter of a cultureliquid (g/L), and the X axis shows the cultivation time (h). The soliddiamond shows the result at 32° C., the open square shows the result at37° C., the open triangle shows the result at 41° C., the mark x showsthe result at 44° C., the solid circle shows the result at 47° C., andthe solid triangle shows the result at 50° C.

FIG. 1B shows the effect of the temperature on the growth of Clostridiumperfringens strain HN001. FIG. 1B particularly shows the calculationresults of the maximum growth rates at respective temperatures.

FIG. 2A shows the hydrogen yields at respective temperatures in Example1.

FIG. 2B shows the maximum hydrogen production rates per liter of theculture liquid at respective temperatures.

BEST MODE FOR CARRYING OUT THE INVENTION

The YNU anaerobic culture medium of the present invention consists of acomposition shown in Table 1, and is suitable for cultivation ofanaerobes. The respective reagents are commercially available in usualmarkets. For example, the followings can be used: casein peptonemanufactured by Nihon Pharmaceutical, Dried Yeast Extract-S manufacturedby Wako Pure Chemical, L-cystein hydrochloride monohydrate manufacturedby Junsei Chemical, mercaptoacetic acid manufactured by Junsei Chemical,and D(+) glucose manufactured by Wako Pure Chemical. The glucoseconcentration can be appropriately determined according to thecultivation condition or the like. In addition, casamino acid may beused instead of casein peptone.

TABLE 1 Composition of YNU anaerobic culture medium (g/L) Casein peptone25.0 Dried Yeast Extract-S 22.0 L-cystein hydrochloride monohydrate 0.3Mercaptoacetic acid 0.3 D(+) glucose 15.0

The bacteria belonging to the genus Clostridium of the presentinvention, in particular Clostridium perfringens, are hydrogen-producingbacteria which have a property of producing hydrogen at a rate of 60mmol or more per hour per liter of a culture liquid which containsglucose as a substrate, by batch cultivation in a YNU anaerobic culturemedium at 47° C. and pH 6.0. The reason is that the hydrogen productionat about 50° C. leads to a higher reaction rate of the hydrogenfermentation than at 30 to 38° C., and thus can be considered tocontribute to more efficient hydrogen production. Another reason is thata hydrogen-producing bacterium exhibiting excellent hydrogenproductivity which achieves a hydrogen production rate of 60 mmol ormore per hour per liter of a culture liquid, is capable of producing alarger amount of hydrogen than ever before, even from biomass containinga wide variety of substances as a production source. The hydrogenproductivity is not specifically limited as long as it achieves ahydrogen production rate of 60 mmol or more, more preferably 80 mmol ormore, and particularly preferably 100 mmol or more, per hour per literof a culture liquid.

The bacterium belonging to the genus Clostridium of the presentinvention can be acquired by the following manner, for example. First,bacterial strains are isolated from nature-derived samples. From thesebacterial strains is selected a bacterial strain exhibiting a hydrogenproductivity which achieves a hydrogen production rate of 60 mmol ormore per hour per liter of a culture liquid at about 50° C., by whichthe bacterium belonging to the genus Clostridium of the presentinvention can be acquired.

Hereunder is a more detailed description.

1. Acquisition of Bacterial Strain

Samples including liquids and sludges collected from sewage and the likewere inoculated in an ABCM semisolid stab culture medium having acomposition shown in Table 2 (Eiken Chemical), and then incubated in athermostat at 50° C., followed by selection of samples exhibiting activegas productions. The thus selected samples were applied to an ABCM agarmedium (Eiken Chemical), and subjected to anaerobic cultivation at 50°C. to thereby obtain colonies. Further, from these colonies, bacteriawere aseptically taken and subjected to anaerobic cultivation in thesame manner to thereby acquire purely isolated bacterial strains. Thepurely isolated bacterial strains were inoculated in the YNU anaerobicculture medium and further incubated in the thermostat at 50° C. tothereby select samples exhibiting active gas productions. From the thusselected bacterial strains, bacterial strains exhibiting hydrogenproductivities which achieved hydrogen production rates of 60 mmol ormore per hour per liter of the culture liquid were selected. Of these,one strain was named HN001 and the hydrogen productivity thereof wasexamined more in detail. The hydrogen production rate was measured bythe method of Example 1 that will be described later.

TABLE 2 Composition of ABCM semisolid medium (g/L) Plant extract 2.0Yeast extract 5.0 Meat extract 3.0 Peptone 10.0 Tryptone 10.0 Soypeptone3.0 Soluble starch 5.0 Glucose 3.0 Sodium chloride 2.0 Dipotassiumphosphate 2.5 L-cysteine hydrochloride 0.3 Sodium thioglycolate 0.3Hemin 0.005 Agar 2.0

2. Identification of Strain 1-1N001 and its Biochemical Character

In order to investigate the genetic property of the strain HN001, the16S rDNA sequence of the strain HN001 was identified by a usual method.The 16S rDNA sequence is shown in SEQ ID: 1 in the sequence listing.This nucleotide sequence was subjected to the homology search on theinternational nucleotide sequence database (GenBank/DDJ/EMBL), theresult of which showed a 98% homology with the nucleotide sequence ofClostridium perfringens ATCC 13124. Accordingly, the strain HN001assumably belongs to Clostridium perfringens.

Furthermore, the biochemical character of the strain HN001 wasinvestigated using the identification kit for strict anaerobes, API20Asystem (manufactured by bioMerieux S.A., imported and distributed bybioMerieux Japan) according to the instruction of the manufacturer.Specifically, a bacterial suspension of the strain HN001 was dispensedin tubes containing respective substrates, and anaerobically incubatedfor 24 hours. Then, based on the color change of the respective tubes,sugar-decomposing abilities and the like of the strain HN001 weredetermined by using the APILAB software (manufactured by bioMerieuxS.A.).

TABLE 3 Substrate Bacteria MD URE GLU MAN LAC SAC MAL SAL XYL ARA GELESC HN001 0 0 99 0 0 99 99 0 0 0 40 20 C. perfringens 0 0 99 0 88 91 991 0 0 99 1 Acti. viscosus 0 0 99 0 64 99 99 21 0 0 14 0 Substrate GLYCEL MNE MLZ RAF SOR RHA TRE HN001 75 0 99 0 75 0 0 0 C. perfringens 79 499 0 16 16 0 75 Acti. viscosus 62 21 96 0 94 0 0 7

Table 3 is a summary of the analysis results by the APILAB software fromfive times independent experiments using the API20A system. The tablealso shows the expected measurement results of respective bacterialstrains. The top lines in the table show the substrate contained in eachtube. IND represents tryptophane, URE represents urea, GLU representsglucose, MAN represents mannite, LAC represents lactose, SAC representssucrose, MAL represents maltose, SAL represents salicin, XYL representsxylose, ARA represents arabinose, GEL represents gelatin, ESC representsesculin ferric citrate, GLY represents glycerin, CEL representscellobiose, MNE represents mannose, MLZ represents melezitose, RAFrepresents raffinose, SOR represents sorbitol, RHA represents rhamnose,and TRE represents trehalose.

As a result, the strain HN001 exhibited a glucose-decomposing ability, asucrose-decomposing ability, a maltose-decomposing ability, and amannose-decomposing ability, like Clostridium perfringens. On the otherhand, unlike the known Clostridium perfringens, the strain HN001exhibited a raffinose-decomposing ability while exhibiting neitherlactose-decomposing ability nor trehalose-decomposing ability. In thedetermination by the APILAB software, the strain HN001 was concluded tobe closer to Actinomyces viscosus than to Clostridium perfringens.

From its genetic property, the strain HN001 was confirmed to be abacterium belonging to Clostridium perfringens. In addition, from thefact that the homology was 98%, and no known bacterium having a completehomology was found, and the biochemical character exhibited araffinose-decomposing ability while exhibiting neitherlactose-decomposing ability nor trehalose-decomposing ability, it isapparent that the strain HN001 has different properties to those ofknown bacterial strains.

Therefore, the applicants deposited the strain HN001 with the NationalInstitute of Technology and Evaluation, Patent Microorganisms Depositary(2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, #292-0818, Japan) as anational deposit (accession number: NITE P-318 original deposit) on Feb.23, 2007. Furthermore, the applicants made a request for transfer to theinternational deposit under the Budapest Treaty, to the concernedinternational depositary authority on Mar. 3, 2008, and a receipt inrespect of the original deposit (accession number of NITE BP-318) wasissued on Mar. 7, 2008,

3. Optimum Temperature for Growth

In order to investigate the effect of temperature on the growth ofClostridium perfringens strain HN001, cultivation was carried out atrespective temperatures of 32, 37, 41, 44, 47, and 50° C.

Specifically, first, Clostridium perfringens strain HN001 was inoculatedin a test tube containing an ABCM semisolid stab culture medium at pH6.0, and was subjected to anaerobic cultivation at 30° C. for 16 hoursas a preparation of a pre-culture liquid. The pre-culture liquid wasadded at 8 mL each to 350 mL of the YNU anaerobic culture medium at pH6.0, followed by cultivation under the respective temperatures, andobservation of the timewise change regarding the bacterial amount in theculture liquid. The cultivation was carried out at a stirring speed of30 rpm. In addition, the bacterial amount in the culture liquid wasmeasured by the following manner: each culture liquid was centrifuged,the bacterial matter was collected in a form of precipitate, and the dryweight of the precipitate was measured.

FIG. 1 shows the effect of the temperature on the growth of Clostridiumperfringens strain HN001. FIG. 1A shows the measurement results oftimewise changes in the bacterial dry weight at the respectivetemperatures, wherein the Y axis shows the bacterial dry weight perliter of the culture liquid (g/L), and the X axis shows the cultivationtime (h). The solid diamond shows the result at 32° C., the open squareshows the result at 37° C., the open triangle shows the result at 41°C., the mark x shows the result at 44° C., the solid circle shows theresult at 47° C., and the solid triangle shows the result at 50° C. FIG.1B shows the calculation results of the maximum growth rates at therespective temperatures obtained based on the measurement results. Fromthese results it is apparent that the preferable growth temperature ofClostridium perfringens strain HN001 is 44 to 47° C.

The growth of Clostridium perfringens strain HN001 was confirmed at 51°C., but not at 53° C. From these results, Clostridium perfringens strainHN001 was found to be growable at 32° C. to 51° C.

4. Measurement of Hydrogen Productivity

Clostridium perfringens strain HN001 was inoculated in a test tubecontaining 16 mL of the ABCM semisolid stab culture medium, and wassubjected to anaerobic cultivation at 30° C. for 16 hours. Then, inorder to acclimatize to high temperatures, the test tube wasanaerobically incubated at 45° C. for 30 minutes for use as apre-culture liquid. The pre-culture liquid was inoculated respectivelyin the YNU anaerobic culture medium at pH 6.0, or a YNU anaerobicculture medium at pH 6.0 which had been prepared to contain glucose at aconcentration of 2.0%. Then, batch cultivation was carried out at 47° C.in the same manner as that of Example 1 that will be described later, toproduce hydrogen. The amount of the produced hydrogen was measured bythe method of Example 1 that will be described later, followed bycalculation of the hydrogen yield (mol/mol-monosaccharide) and themaximum hydrogen production rate. Here, the term hydrogen yield refersto a number of moles of hydrogen produced from 1 mol of monosaccharide.Moreover, the maximum hydrogen production rate was expressed by twokinds of denotations: a maximum hydrogen production rate per liter ofthe culture liquid (mmol/Lh); and a maximum hydrogen production rate pergram of the bacterial dry weight in the culture liquid (mmol/gh).

The obtained hydrogen yield and the maximum hydrogen production rate ofthe Clostridium perfringens strain HN001 were compared with those ofknown representative hydrogen-producing bacteria. The results of thiscomparison are shown in Table 4. The hydrogen yield and the like ofthese bacterial strains except for the strain HN001 are based on thedescription of Non-patent Document 1. The parts having no description ofthe corresponding data in the document are left with bars.

TABLE 4 Cultivation Temp. Hydrogen yield Hydrogen production rate methodpH [° C.] Substrate [mol/mol] [mmol/L · h] [mmol/g · h] C. perfringens B6 47 glucose 2.4 160 44 HN001 (2.0%) C. perfringens B 6 47 glucose 2.2135 37 HN001 (1.5%) Bacteria belonging to Clostridium C. beijerinckii B— 41 glucose 2 24 25 AM21B C. paraputrificum B — 37 GlcNAc 2.5 31 — M-21C. butyricum LMG C 5.8 36 glucose 1.5 22 — 1213tl Clostridium sp. No 2 C6 36 glucose 2.4 21 — Bacteria belonging to Enterobacter E. aerogenesE.82005 B 6 38 glucose 1 21 17 E. cloacae IIT-BT 08 B — 36 sucrose 3 3529 E. aerogenes E.2005 C 6 38 molasses 0.7 36 17 E. aerogenes C — 37glucose 1.1 58 — HU-101mAY-2 High-temperature bacteria Thermotogamaritima B — 80 glucose 4 10 — Thermotoga elfii B 7.4 65 glucose 3.3 3 5Caldicellulosiruptor B 7 70 sucrose 3.3 8 12 saccharolyticus Clostridiumthermocellum B — 60 cellobiose 1 7 14 B: Batch cultivation, C:Continuous cultivation, Unit of hydrogen yield is[mol/mol-monosaccharide].

Clostridium perfringens strain HN001 showed a hydrogen yield of 2.4 bybatch cultivation at 47° C. and pH 6.0. That is, the bacterial strainwas capable of producing 2.4 mol of hydrogen from 1 mol of glucose. Inaddition, the maximum hydrogen production rate per liter of the cultureliquid was 160 mmol/Lh, and the maximum hydrogen production rate pergram of the bacterial dry weight in the culture liquid was 44 mmol/gh.

On the other hand, as for the other bacterial strains described in thetable, the highest maximum hydrogen production rate per liter of theculture liquid was 58 mmol/Lh of Enterobacter aerogenes HU-101 mAY-2,and the highest maximum hydrogen production rate per gram of thebacterial dry weight in the culture liquid was 29 mmol/Lh ofEnterobacter cloacae IIT-BT08. Since the comparison was made withreference values obtained under different experimental conditions, nosimple comparison could be made. However, it is apparent thatClostridium perfringens strain HN001 has a much higher maximum hydrogenproduction rate than those of other known hydrogen-producing bacteria.Moreover, the hydrogen yield was sufficiently satisfactory. Accordingly,it is apparent that Clostridium perfringens strain HN001, that is, thebacterium belonging to the genus Clostridium of the present inventionexcels in hydrogen productivity much better than the other knownhydrogen-producing bacteria.

From the abovementioned results, as for the bacterium belonging to thegenus Clostridium of the present invention, in particular, Clostridiumperfringens, there is employed a hydrogen-producing bacterium which hasa property of producing hydrogen at a rate of 60 mmol or more per hourper liter of a culture liquid which contains glucose as a substrate, bybatch cultivation in the YNU anaerobic culture medium at 47° C. and pH6.0. Furthermore, the hydrogen productivity of the bacterium belongingto the genus Clostridium of the present invention preferably achieves ahydrogen production rate of 80 mmol or more, and particularly preferably100 mmol or more, per hour per liter of the culture liquid. The use ofthe bacterium belonging to the genus Clostridium having a hydrogenproductivity within this range enables hydrogen production with higherefficiency than ever before.

In addition, the upper limit of the hydrogen production rate of thebacterium belonging to the genus Clostridium of the present invention isnot specifically limited as long as it is 60 mmol or more per hour perliter of a culture liquid under the abovementioned cultivationcondition, although the hydrogen production rate is preferably higher.The bacteria belonging to the genus Clostridium of the present inventionacquired by the abovementioned method normally have hydrogen productionrates of 60 to 250 mmol under the same condition. Moreover, thepreferred range of the hydrogen production rate is 80 to 200 mmol, morepreferably 100 to 180 mmol, and most preferably 100 to 160 mmol.

5. Optimum Temperature for Hydrogen Production

The pre-culture liquid was prepared in the same manner as theabove-mentioned paragraph 4. Measurement of hydrogen productivity. Thepre-culture liquid was added at 8 mL each to 350 mL of the YNU anaerobicculture medium at pH 6.0 or 6.5 in a 500 mL volume flask, followed bybatch cultivation at a stirring speed of 30 rpm in the same manner asthat of Example 1 that will be described later, to produce hydrogen. Thecultivation temperature was set at respectively 41, 44, 47, and 50° C.Moreover, the pH was kept constant by an automatic controller. Theamount of the produced hydrogen was measured by the method of Example 1that will be described later, followed by calculation of the hydrogenyield and the maximum hydrogen production rate per liter of the cultureliquid.

TABLE 5 Cultivation Hydrogen production Hydrogen yield temperature [°C.] rate [mmol/L · h] [mol/mol-glucose] pH 6.0 41 59.8 1.30 44 94.2 1.9347 119.2 2.15 50 111.1 2.22 pH 6.5 41 41.1 0.82 44 71.4 1.39 47 91.11.42 50 80.4 2.12 Glucose concentration: 1.5%

Table 5 shows the hydrogen yields and the maximum hydrogen productionrates per liter of the culture liquid at respective cultivationtemperatures. The maximum hydrogen production rate showed the lowestvalues at 41° C. for both pH 6.0 and pH 6.5, which were respectively59.8 mmol/Lh and 41.1 mmol/Lh. These values are lower than the maximumhydrogen production rates at other cultivation temperatures, but aresufficiently satisfactory maximum hydrogen production rates as isapparent from a comparison with Table 4. Accordingly, it is apparentthat Clostridium perfringens strain HN001 is capable of satisfactorilyproducing hydrogen at 47 to 50° C.

In addition, as is apparent from Table 5, the maximum hydrogenproduction rate was highest at 47° C. for both pH 6.0 and pH 6.5, andthe hydrogen yield was highest at 50° C. for both pH 6.0 and pH 6.5.That is to say, it is apparent that the most suitable temperature forthe hydrogen production of Clostridium perfringens strain HN001, thatis, the bacterium belonging to the genus Clostridium of the presentinvention is 47° C. to 50° C. In addition, since the optimum temperaturefor the growth is 44 to 47° C., the growth rate was found to be notnecessarily proportional to the hydrogen production rate.

The bacterium belonging to the genus Clostridium of the presentinvention was acquired from bacteria which have originally been residingin liquids and sludges collected from sewage and the like, namelybiomass, through selection of a bacterium which excels in hydrogenproductivity, in particular, hydrogen production rate. Accordingly, thebacterium belonging to the genus Clostridium of the present invention iscapable of producing a large amount of hydrogen with higher efficiencythan ever before, even from biomass as a production source.

The bacterium belonging to the genus Clostridium of the presentinvention can be cultured by a usual method for use in the cultivationof bacteria belonging to the genus Clostridium, and anaerobiccultivation is preferred. Either batch cultivation or continuouscultivation may be employed. Since the risk of contamination can bereduced and no special devices are needed, batch cultivation ispreferred for the production of a small amount of hydrogen. On the otherhand, since the cultivation condition can be readily kept constant andthe productivity can be stabilized, continuous cultivation is preferredfor the production of a large amount of hydrogen such as industrialproduction. When performing continuous cultivation, the bacteriumbelonging to the genus Clostridium of the present invention may also beimmobilized to a usual carrier.

The culture liquid for use in the cultivation of the bacterium belongingto the genus Clostridium of the present invention is not specificallylimited, and usual culture liquids for use in cultivation of bacteriabelonging to the genus Clostridium including commercially availablemedia for anaerobes can be used. Examples of these media include theABCM semisolid medium and the YNU anaerobic culture medium. The glucoseconcentration of the culture liquid can be appropriately determinedaccording to the cultivation condition or the like. In addition, thecultivation temperature is not specifically limited as long as thebacterium belonging to the genus Clostridium of the present inventioncan grow, although preferred is 44 to 47° C.

Efficient hydrogen production can be achieved by cultivation of thebacterium belonging to the genus Clostridium of the present invention bya usual method. The culture liquid for the hydrogen production is notspecifically limited as long as it is a usual culture liquid for use incultivation of bacteria belonging to the genus Clostridium, although theYNU anaerobic culture medium is preferred. In addition, a food wastesuch as raw garbage and other industrial wastes may also be used as theraw material of the culture liquid for the bacterium belonging to thegenus Clostridium of the present invention as long as the effect of thepresent invention is not impaired. Even from such a raw material, thebacterium belonging to the genus Clostridium of the present inventioncan enable hydrogen production with higher efficiency than ever before.

In addition, the cultivation temperature for the hydrogen production ispreferably 47 to 50° C. from the viewpoint of hydrogen productionefficiency. Moreover, the pH of the culture liquid for the hydrogenproduction is not specifically limited, although it is preferably 5.8 to6.5, and particularly preferably 6.0 to 6.2.

Moreover, the sugar component contained in the culture liquid or thefood wastes etc. as the fermentation medium for the hydrogen productionis not specifically limited as long as it can serve as the substrate forthe hydrogen fermentation of the bacterium belonging to the genusClostridium of the present invention. Examples thereof can includemonosaccharides such as glucose, maltose, and sucrose, andpolysaccharides such as starch. The concentration of such componentsserving as the substrate for the hydrogen fermentation can beappropriately determined according to the cultivation condition or thelike, although it is preferably 0.5 to 5% by weight, and more preferably1.5 to 2.5% by weight in the culture liquid.

Furthermore, other cultivation conditions for the hydrogen productionare not specifically limited as long as the effect of the presentinvention is not impaired. However, the bacterium belonging to the genusClostridium of the present invention is preferably cultured at ahydrogen production rate within a range of 60 to 250 mmol/L·h,preferably 80 to 200 mmol/L·h, and more preferably 100 to 180 mmol/L·h,as well as the abovementioned conditions of the cultivation temperature,the culture liquid, and the like.

Next is a more detailed description of the present invention withexamples. However, the present invention is in no way limited to thefollowing examples.

EXAMPLE 1

Clostridium perfringens strain HN001 was inoculated in a test tubecontaining 16 n L of the ABCM semisolid stab culture medium, and wassubjected to anaerobic cultivation at 30° C. for 16 hours. Then, inorder to acclimatize to high temperatures, the test tube wasanaerobically incubated at 45° C. for 30 minutes for use as apre-culture liquid. The pre-culture liquid was added at 8 mL each to 350mL of the YNU anaerobic culture medium at pH 6.0 in a 500 mL jarfermenter, followed by batch cultivation at a stirring speed of 30 rpm.The cultivation temperature was set at 32, 37, 41, 44, 47, and 50° C.,respectively. Moreover, the pH was kept constant by an automaticcontroller.

The produced hydrogen gas was collected in a measuring cylinder andquantified by the liquid displacement method using 10% sodium hydroxideaqueous solution to remove carbon dioxide. The thus collected gas wasconfirmed to be hydrogen gas through analysis using gas chromatography.The amount of the produced amount was used to calculate the hydrogenyield and the maximum hydrogen production rate per liter of the cultureliquid.

FIG. 2A shows the hydrogen yields at the respective cultivationtemperatures and FIG. 2B shows the maximum hydrogen production rates perliter of the culture liquid at the respective cultivation temperatures.The maximum hydrogen production rate at 47° C. was about 135 mmol/Lh. Inaddition, the maximum hydrogen production rate was highest at 47° C.,while the hydrogen yield was found to be prone to increase at about 32°C. and 50° C.

From the results of Example 1, it is apparent that Clostridiumperfringens strain HN001, that is, the bacterium belonging to the genusClostridium of the present invention is capable of producing hydrogen ata rate of 60 mmol or more per hour per liter of a culture liquid whichcontains glucose as a substrate, by batch cultivation in the YNUanaerobic culture medium at 47° C. and pH 6.0, and the most suitablecultivation temperature for the hydrogen production is 47° C. to 50° C.

EXAMPLE 2

The amount of hydrogen produced from each culture was measured in thesame manner as that of Example 1, except that the pH of the YNUanaerobic culture medium was set at 5.5, 6.0, 6.5, and 7.0, and thecultivation temperature was set at 47° C. only. Then, the hydrogen yieldand the maximum hydrogen production rate per liter of the culture liquidwere calculated.

TABLE 6 Hydrogen production Hydrogen yield pH for cultivation rate[mmol/L · h] [mol/mol-glucose] 5.5 33.9 1.96 6.0 111.2 2.22 6.5 80.42.12 7.0 38.4 1.29 Glucose concentration: 1.5%

Table 6 shows the hydrogen yields and the maximum hydrogen productionrates per liter of the culture liquid at respective pH values. Thehydrogen yield and the maximum hydrogen production rate were bothhighest at 6.0. The maximum hydrogen production rate at pH 6.5 was 80.4mmol/Lh which greatly exceeded 60 mmol/1 Therefore it is apparent thatthe pH of the culture liquid for the hydrogen production is preferably5.8 to 6.5, and particularly preferably 6.0 to 6.2.

INDUSTRIAL APPLICABILITY

The bacterium belonging to the genus Clostridium of the presentinvention excels in hydrogen productivity, and thus is usable in thefield of hydrogen production from biomass as a production source.

1. An isolated bacterium Clostridium perfringens, which has a propertyof producing hydrogen at a rate of 60 mmol or more per hour per liter ofa culture liquid which contains glucose as a substrate, by batchcultivation in a YNU anaerobic culture medium at 47° C. and pH 6.0,wherein the bacterial strain is Clostridium perfringens HN001 (NITEBP-318).
 2. An isolated bacterium Clostridium perfingens, according toclaim 1, wherein the optimum temperature for hydrogen production is 47to 50° C.
 3. An isolated bacterium Clostridium perfringens, according toclaim 1, wherein the optimum temperature for cell growth is 44 to 47° C.4. An isolated bacterium Clostridium perfringens, according to claim 1,wherein the bacterium has a raffinose-decomposing ability.
 5. A methodof producing hydrogen comprising: anaerobically incubating the isolatedbacterium Clostridium perfringens according to claim 1 at a pH of 6.0 to6.5 and at a temperature of 45 to 50° C.
 6. A method of producinghydrogen according to claim 5, wherein the temperature is 47 to 50° C.